Anti-human hepatoma monoclonal antibody hav18 light/heavy chain available region gene, and use thereof

ABSTRACT

The present invention relates to the anti-human hepatocarcinoma McAb HAb18 heavy chain and light chain variable region genes, the polypeptides encoded by the same, as well as to their use in the preparation of a medicament for diagnosing and treating tumors or inflammation diseases. Based on the heavy chain and light chain variable region genes, various novel small molecule genetic engineering antibodies, including single chain antibodies, chimeric antibodies, Fab antibodies and the like can be constructed and expressed for the diagnosis and treatment of hepatoma.

FIELD OF THE INVENTION

This invention relates to the anti-human hepatoma monoclonal antibody(McAb) HAb18 heavy/light chain variable region genes and the codedpolypeptides thereof, and to the use of the genes and polypeptides inthe preparation of medicaments for diagnosing and treating tumors orinflammatory diseases.

BACKGROUND OF THE INVENTION

Hepatoma is a common disease with high morbidity in China and SoutheastAsia. Particularly in China, about 110,000 patients die of hepatoma eachyear, which holds 43% among the total dead hepatoma patients in theworld. Therefore, it still requires comprehensive and profound researchon hepatoma diagnosis and therapy. A variety of McAbs have beenidentified since hybridoma technology was established by Kohler andMilstein in 1975. These McAbs shed new light on the preclinical andclinical research and anti-hepatoma McAbs are no exception. In the past20 years, traditional production of anti-hepatoma antibodies has focusedon such target molecules as α-fetoprotein and ferritin. In theseprocesses, traditional immunization methods are adopted, i.e. with thesoluble extraction materials of hepatocarcinoma or culturedhepatocarcinoma cells as the immunogen. However, it is difficult togenerate anti-hepatoma antibodies with high affinity and specificity bythese methods because of the variation, instability or partial loss ofthe tumor antigenic epitopes.

There are several successful reports on antibody generation using cellsuspensions derived from fresh tumor tissue to immunize animals. Inspite of the complicated screening, antigenicity can be better retainedin this way. The inventors have successfully obtained seven clones ofspecific anti-hepatoma antibodies using this method. The detailedprocess for the establishment of these hybridoma cell strains thatsecret murine anti-hepatoma antibodies is shown in Zhinan Chen, YanfangLiu, Jizheng Yang et al., McAb Communications, 1989; 2: 33-36. Amongthese antibodies, anti-human hepatoma McAb HAb18 was found to havehigher specificity, with an immunohistochemistry positive rate up to75%. Starting from anti-human hepatoma McAb HAb18, the inventors havesuccessfully cloned heavy and light chain variable region genes. Withthe advances of the research of genetically engineered antibody, thisinvention establishes a solid basis to develop novel small moleculegenetically engineered antibody.

SUMMARY OF THE INVENTION

In one aspect, the invention relates to the variable region genes of themonoclonal antibody HAb18 against hepatoma-associated antigen HAb18G.These McAb HAb18 variable region genes were cloned from a hybridoma cellline HAb18 that can secret murine anti-HAb18G monoclonal antibodies withhigh activity. The full length of the heavy chain variable region geneis 445 bp, with a nucleic acid sequence shown in SEQ ID NO: 1. The aminoacid sequence encoded by this gene is shown in SEQ ID NO: 3. The fulllength of the light chain variable region gene is 421 bp, with a nucleicacid shown in SEQ ID NO: 2. The amino acid sequence encoded by this geneis set forth in SEQ ID NO: 4. After recombination, these two genes maybe used to express active antibody fragments which can specificallyidentify and bind the hepatoma-associated antigen HAb18G. It iscontemplated that the invention also comprises those sequences that canhybridize with the sequences in SEQ ID NO: 1 or SEQ ID NO: 2 understringent hybridization conditions, for example, the conditions statedin Molecular Cloning (Cold Spring Harbor Laboratory).

In another aspect, the invention relates to a Fab gene comprising theheavy chain and the light chain variable region genes, a murine-humanchimeric Fab gene, as well as the gene products thereof, namely a Fabantibody and chimeric Fab antibody.

In still another aspect, the invention relates to the use of the genesand polypeptides of the invention in the preparation of medicaments fordiagnosing and treating tumors or inflammation diseases.

DETAILED DESCRIPTION OF THE INVENTION

Using a set of designed primers, the inventors have cloned the McAbHAb18 heavy chain and light chain variable region genes from a hybridomacell line HAb18 that can secret specific monoclonal antibodies againsthepatoma. Sequence analysis shows that the heavy chain variable regiongene (VH gene) is highly homologous with the variable region gene of theautoantibody against thymocytes and erythrocytes described by Kasturi,K. N et al. The light chain variable region gene (VL gene) belongs tothe V-J region gene of the rearranged κ chain of mouse immunoglobulin,and has highest homology with the anti-DNA antibody 6E6 light chainvariable region gene, which was described by O'Connor, K. C et al. TheVH and VL genes of the invention encode accurate variable regions of themouse antibody. With these genes, a variety of novel small moleculeantibodies, including single chain antibody, chimeric antibody, Fabantibody, and the like, may be constructed and expressed by geneticengineering for the diagnosis and therapy of hepatoma.

So far, monoclonal antibodies against hepatoma are mainly used inresearch on hepatocarcinoma associated antigens. The development ofmonoclonal antibodies provides an effective way to discover novel tumorassociated antigens or new target sites. These antigens can be used inclinical serum tests and are helpful in diagnosing and evaluatingtherapeutic efficacy. Monoclonal antibodies can also be used in researchon hepatoma monoclonal-antibody-targeted drugs. Radioimmunodiagnosis,targeted chemotherapy, and radiotherapy with McAbs as carriers have madesubstantial progress in recent years. In this regard, drugs such aschemical compound-McAb conjugates, cytotoxin-McAb conjugates andradionuclide-McAb conjugates have been developed.

The light and heavy chain variable region genes of the invention can beused to construct certain protein medicaments, which can be directlyused in the diagnosis and treatment of relevant diseases, especiallytumors and inflammation diseases. The polypeptide encoded by the genesof the invention can be conjugated with a cytotoxin, a toxin, aradionuclide, an enzyme or a biological response modifier, so as to beused as targeted drugs in the diagnosis and therapy of certain diseases,especially tumors.

From the polypeptides encoded by the genes of this invention, new typesof antibodies such as chimeric antibodies, humanized antibodies, smallmolecule antibodies, multi-covalent miniantibodies, bispecificantibodies, recombinant antibody fusion proteins, recombinantimmunotoxins and phage antibodies may be constructed.

For a chimeric antibody, the variable region of a mouse McAb may belinked with the constant region of a human Ig. Desirable efficacies intumor therapy have been achieved by using such chimeric antibodiesbecause these antibodies have reduced side effects such as the HAMAresponse while maintaining the specificity and affinity of a mouse McAb.

To obtain a humanized antibody, the structure of variable region genesis humanized by means of CDR graft, surface amino veneer, frameworkexchange, located reservation and epitope-directed selection. Ahumanized antibody can keep the specificity and affinity of a mouseMcAb.

Small molecule antibodies include Fab antibodies consisting of VH-CH1and VL-C1, single chain antibodies formed by linking VH and VL geneswith a polypeptide linker (Gly4Ser)₃, Fv fragment antibodies formed bynon-covalent linkage of VH and VL, single domain antibodies comprisingonly one domain of VH or VL, and minimal recognition units constructedby single CDR, and the like.

Multi-covalent miniantibodies mainly include double chain antibodies,(ScFv)₂, Flex mini-antibodies, LD mini-antibodies, F(ab′)₂, F(ab′)₃,(ScFv)₄, etc. The multi-covalent miniantibodies have good potentialvalue in clinical applications because they have multiple antigenbinding sites, high affinity, moderate molecule size, high ability topenetrate tumor tissues, and slow clearance rate in kidney.

Bispecific antibodies, also called bifunctional antibodies, have doublespecificities and functions.

Recombinant antibody fusion proteins are recombinant proteins capable oftargeting a specific biological activity at a target site, and areprepared by linking a Fab or Fv gene with a gene encoding a non-antibodyprotein such as a toxin or an enzyme.

Recombinant immunotoxins are produced by linking a gene for a toxin anda gene for an antibody and expressing the resultant gene. They have lownon-specificity, are stable and safe in vivo and easily penetrate intotumors.

A phage antibody is obtained by linking an Ig V gene with a phage geneIII or gene VIII, then the gene is transfected into host bacteria, whichexpresses the fusion protein of Fab or ScFv on the surface of a phagemembrane. Through several affinity pannings, specific antibodies can beobtained.

Using the polypeptides encoded by the heavy chain and light chainvariable region genes of the invention as a carrier, various anti-canceror anti-inflammation agents may be conjugated with said polypeptides soas to form immunoconjugates or targeted drugs. For example, theimmunoconjugates or targeted drugs can be in the form of a conjugate ofan antibody and a nuclide. Such a conjugate is able to direct thenuclide efficiently to local tumor tissues, reducing the damage tonormal tissues incurred by irradiation during radiotherapy. Theconjugate also allows a targeted diagnosis and treatment of tumors,termed radioimmunoimaging and radioimmunotherapy respectively. Nuclidesnormally used to conjugate with a monoclonal antibody include ¹²⁵I,¹³¹I, ¹¹¹In, ⁹⁰Y, ⁹⁹Tc^(m), ¹⁸⁸Re and ¹⁸⁶Re, and the like.

The immunoconjugates or targeted drugs can also be in the form of aconjugate of an antibody and a chemotherapy drug. Such a conjugatedirects the drug specifically to tumors, reducing the damage to normaltissues and the side effects caused by chemotherapy drugs. Chemotherapydrugs commonly used to conjugate with antibodies include alkylatingagents such as phosphoramides, anti-metabolites such as methotrexate and5-flurouracil, antibiotics such as adriblastine, epirubicin, cerubidin,leurocristine and mitomycin.

The immunoconjugates or targeted drugs can also be in a form of aconjugate of an antibody and a toxin, which is also called animmunotoxin. Immunotoxins have strong cytotoxicity and are independentfrom biological auxiliary mechanisms. Because immunotoxins have amechanism different from traditional chemotherapy and radiotherapy forkilling tumors, they can be used to treat tumors on which chemotherapyand radiotherapy have poor effects. Toxins that may be used to conjugatewith an antibody include ricin, diphtheria toxin, ormosia toxin,soapwort, pseudomonas exotoxin, streptolysin, porforin, etc.

The immunoconjugates or targeted drugs can also be in the form of aconjugate of an antibody and a biological response modifier (BRM).Although BRMs alone can adjust the immune function of human body andkill tumor cells with good efficiency, its function is not exertedcompletely because only a part of the BRM injected can arrive at thetarget site. BRMs also have some side effects. If a BRM is conjugatedwith a McAb, the McAb can guide the BRM to the target site and killtumor cells. BRM includes INF and IL-2.

The immunoconjugates or targeted drugs can also be in the form of anantibody-targeted prodrug. Antibodies are conjugated with an enzyme thatcan specifically activate prodrugs. The conjugate is first injected intothe body, after some time, a prodrug is injected, which can be convertedinto an active drug at tumor site and kill tumor cells. At present, somecompounds can be used as prodrugs, including glutamine derivatives ofbenzoic acid hydrogen mustard, phosphoric acid podophyllum ethylidene,phosphoric acid mitocin-C, glycoside cerubidin, adriblastine,5-fluorocytosine and cephalothin chlorethazine. The activating enzymesinclude carboxypeptidase G₂, alkaline phosphatase, penicillin amidase,β-lactamase, cytosine deaminase, β-glycosidase, and aminopeptidase.

The immunoconjugates or targeted drugs can also be in the form of animmune liposome, in which a McAb is conjugated to the surface of aliposome. Because the liposome can encapsulate large amounts of drugsand the antibody can bind antigen specifically, immune liposomes withencapsulated drugs have improved specificity and efficacy.

DESCRIPTION OF THE FIGURES AND PREFERRED EMBODIMENTS

The invention is further described with reference to the followingfigures and examples.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Agarose gel electrophoresis of the heavy/light chain variableregion genes of anti-human hepatoma McAb HAb18 amplified by RT-PCR. A,DL-2000 marker; B, heavy chain variable region gene of McAb HAb18; C,light chain variable region gene of McAb HAb18.

FIG. 2. Agarose gel electrophoresis analysis of the PCR-amplifiedproduct of the gene for a Fab of anti-human hepatoma McAb HAb18. 1,DL-2000 Marker, 2, Fd gene, 3, light chain gene.

FIG. 3. Construction diagram of the Fab expression vector.

FIG. 4. Restriction analysis of recombinant expression vectorpComb3/Fd-L. 1, pComb3/Fd-L/Xho I+Spe I; 2, pComb3/Fd-L/Sac I+Xba I; 3,pComb3/Fd-gIII-L/Spe I+Nhe I; 4, pComb3/Fd-L/Spe I+Nhe I.

FIG. 5. Competitive ELISA assay for the binding activities of Fab andHAb18 with corresponding antigen.

FIG. 6. Immuno-fluorescence detection of the reactivity of McAb HAb18and Fab fragments thereof with Hepatocarcinoma cell line HHCC. A,Immuno-fluorescence staining using HAb18 Fab; B, Immuno-fluorescencestaining using HAb18.

FIG. 7. Restriction analysis of the recombinant expression vectorpComb3C. M, DNA marker; 1, human IgG3CH1; 2, human κ chain constantregion; 3, pComb3C/XhoI+Spe I; 4, pComb3C/Sac I+Xba I; 5, pComb3C/EcoRV+Sal I.

FIG. 8. Construction of the expression vector containing the chimericFab gene (cFab).

FIG. 9. Restriction analysis of recombinant cFab expression vectorpComb3C/cFab. M, DNA marker; 1, VH; 2, VL; 3, pComb3C/cFab-gIII/XhoI+Spe I; 4, pComb3C/cFab-gIII/EcoRV+Spe I; 5, pComb3C/cFab-gIII/SacI+Xba I; 6, pComb3C/cFab-gIII/Sal I+Xba I; 7, pComb3C/cFab-gIII/SpeI+Nhe I.

FIG. 10. ELISA assay for the binding activity of cFab. 1, supernatant oflysed bacteria transformed with empty vector; 2, supernatant of lysedbacteria transformed with pComb3, before induction; 3, bacteriatransformed with pComb3C/cFab, after induction; 4, chimeric HAb18IgG; 5,PBS.

FIG. 11. Competitive ELISA detection of the binding activities of cFaband HAb18 with corresponding antigen.

FIG. 12. Immuno-fluorescence detection of the reactivity of the chimericHAb18IgG and its cFab with Hepatocarcinoma cell line HHCC. A, Stainingwith HAb18 cFab; B, Staining with chimeric HAb18IgG.

EXAMPLES Example 1 Cloning of the Genes Encoding the Variable Regions ofHeavy/Light Chain of Anti-Human Hepatoma McAb HAb18

The cell line used was a murine hybridoma cell line obtained by using aconventional cell fusion technique by the Cell Engineering ResearchingCenter of the Fourth Military Medical University (FMMU) (Zhinan Chen,Yanfang Liu, Jizheng Yang et al. McAb communication. 1989; 2:33-36). Theantibody produced by the cell line was a murine anti-human hepatoma McAbHAb18, which was an IgG, with high affinity and specificity to itstarget molecule HAb18G.

Cells (5×10⁶) in exponential growth phase were used to prepare totalRNA. Typically, total RNA extraction was carried out using an SV TotalRNA Isolation System according to the protocol provided by themanufacturer (Promega, USA). The quality and quantity of the sample weredetermined by ultraviolet spectroscopy and 1% denaturing polyacrylamidegel electrophoresis. A first strand cDNA corresponding to the variableregion of the antibody was obtained by reverse transcription usingrandom primers Oligo(dT)₁₅ (Promega, USA). The genes encoding thevariable regions of heavy chain and light chain (VH and VL) of murineanti-human hepatoma McAb HAb18 were amplified by PCR with a set ofuniversal primers. The PCR products VH (about 450 bp) and VL (about 421bp) were separated on a 1.5% agarose gel and isolated by cutting thegel, recovered and purified by using a gel purification kit (PromegaInc), and then identified by agarose gel electrophoresis (FIG. 1). Thefragments of interest were cloned respectively into vector pMD-18-T andthen subjected to sequence analysis.

Oligo(dT)₁₅ (Promega, USA) was used as a random primer for reversetranscription. The volume of the reaction system was 20 μL, containing 1μg of total RNA (2 μL), 0.5 μg random primers Oligo(dT)₁₅ (1 μL), 4 μLMgCl₂ (25 mM), 2 μL 5×dNTPs, 2 μL 10× Buffer, 0.5 μL RNase inhibitor.The reverse transcription was carried out in the presence of 0.75 μL ofAMV reverse transcriptase enzyme (15U). RNase-free water was added toreach a total volume of 20 μL. The mixture was incubated at 42° C. for60 min and then boiled for 3 min. The product obtained was then kept at−20° C. until use.

For PCR amplification of VH and VL genes, reactions were carried out ina final reaction volume of 50 μL which contained 5 μL first strand cDNA,30 pmol (5 μL) of reverse and forward primers (the primers are describedbelow), 0.4 mM each of dNTPs, 5 μL 10× buffer and 1.25U Ex Taq DNApolymerase. Water was added to reach a final volume of 50 μL, mixed anda drop of paraffin oil was then added into the mixture. After heated for3 min at 94° C., amplification was performed for 35 cycles with 94° C. 1min, 54° C. 1 min, and 72° C. 1 min for each cycle, and finally theextension reaction was at 72° C. for 10 min.

Cloning of desired fragment into a T vector was conducted as follows.PCR products were recovered on an agarose gel and cloned into pMD18-T.The ligation reaction contained 1 μL of vector pMD18-T, 3 μL of purifiedPCR product (VH or VL gene), 1 μL de-ionized H₂O and 5 μL of ligationbuffer. The reaction system was incubated at 4° C. overnight. Theproduct was transformed into Escherichia coli JM109. Recombinant cloneswere selected and then sequenced using universal primers.

The designed primers used for the amplification of the genes of variableregions of heavy and light chains of McAb HAb18 were as follows(underlined portions represent restriction sites):

5′ Primers for the Variable Region of the Heavy Chain of the MouseAntibody: (1)5′-GGGATATCCACCATGG(AG)ATG(CG)AGCTG(TG)GT(CA)AT(CG)CTCTT-3′ (SEQ IDNO:5) (2) 5′-GGGGATATCCACCATG(AG)ACTTCGGG(TC)TGAGCT(TG)GGTTTT-3′ (SEQ IDNO:6) (3) 5′-GGGGATATCCACCATGGCTGTCTTGGGGCTGCTCTTCT-3′ (SEQ ID NO:7)

3′ Primer for the Variable Region of the Heavy Chain of the MouseAntibody: 5′GAC(ACT)CATGGGG(CG)TGT(TC)GTGCTAGCTG(AC)(AG)GAGAC(AGT)G (SEQID NO:8) TGA-3′

5′ Primers for the Variable Region of the Light Chain of the MouseAntibody: (1) 5′-GGGGATATCCACCATGGAGACAGACACACTCCTGCTAT-3′ (SEQ ID NO:9)(2) 5′-GGGGATATCCACCATGGATTTTCAAGTGCAGATTTTCAG-3′ (SEQ ID NO:10) (3)5′-GGGGATATCCACCATGGAG(AT)CACA(GT)(AT)CTCGGGTCTTT(GA)TA-3′ (SEQ IDNO:11) (4) 5′-GGATATCCACCATG(GT)GCCC(AT)(AG)CTCAG(CT)TC(CT)CT(TG)GT-3′(SEQ ID NO:12) (5) 5′-GGGGATATCCACCATGAAGTTGCCTGTTAGGCTGTTG-3′ (SEQ IDNO:13)

3′ Primer for the Variable Region of the Light Chain of the MouseAntibody: 5′-GGATACAGTTGGTGGTGCAGTCGACTTACGTTT(GT)GTTTCA(AG)CTT-3′ (SEQID NO:14)

The sequences of the genes encoding the variable regions of heavy/lightchain (VH and VL) were referred to as SEQ ID NO:1 and SEQ ID NO:2, andtheir amino acids sequence were referred to as SEQ ID NO:3 and SEQ IDNO:4, respectively.

Example 2 Cloning and Expression of the Gene Encoding Fab of Anti-HumanHepatoma McAb HAb18

The relative molecular weight of Fab is about 50,000. The Fab fragmenthas good penetration and pharmacokinetics properties and has been widelyused in the diagnosis and therapy of many diseases. In this example, thegene encoding the Fab fragment of McAb HAb18 was cloned and expressed inE. coli.

1. Materials and Methods

1.1 Materials

A murine hybridoma cell line producing a murine anti-human hepatoma McAbHAb18 (IgG₁) was obtained by using a conventional cell fusion technique(Zhinan Chen, Yanfang Liu, Jizheng Yang et al. McAb communications.1989; 2:33-36). Trizol reagent (Gibco BRL) and a Reverse Transcriptionkit (Promega, USA) were used. Expression vector pComb3 and E. colicompetent cell JM109 and XL1-blue were commercially obtained. T vector,PCR reagents, restriction endonucleases and ligase were purchased fromTakara (Dalian, China). McAb HAb18 and HRP-HAb18 were made by theinventors (Zhinan Chen, Yanfang Liu, Jizheng Yang et al. McAbcommunications. 1989; 2:33-36). IPTG-, FITC- and HRP-labeled goatanti-mouse IgGs were purchased form SABC Co. A hepatocarcinoma cell lineHHCC was maintained and cultured by the Cell Engineering ResearchingCenter of the FMMU. Primers for PCR were synthesized by Sai Bai ShengBiotech Company (Beijing, China), with the following sequences: Fd5′primer: 5′-AAG TGA AGC TTC TCG AGT CTGG-3′ (SEQ ID NO: 15), Fd3′primer: 5′-AGG CTT ACT AGT ACA ATC CCT GGG CAC AAT-3′ (SEQ ID NO: 16).Light Chain 5′primer: 5′-GAT GTG AGC TCG TGA TGA CCC AGA CTC C-3′ (SEQID NO: 17), Light Chain 3′ primer: 5′-GCG CCG TCTAGA ATT AAC ACT CAT TCCTGT TGA A-3′ (SEQ ID NO: 18). The underlined sequences respectivelyrepresent the restriction sites of Xho I, Spe I, Sac I, and Xba I.

1.2 Methods

1.2.1 Cloning of the Fab Genes of HAb18 McAb:

The total RNA was extracted from the hybridoma cell secreting McAb Hab18 using Trizol reagent and reverse transcribed into cDNA. The cDNA wasused as a template to amplify the Fd and light chain genes. The PCRproducts were purified by agarose gel and separately cloned into pMD18-Tto construct pMD18-T/Fd and pMD18-T/L vectors. After the vectors weretransformed into Escherichia coli JM109, positive clones were screenedand then sequenced.

1.2.2 Construction of the HAb18 Fab Expression Vector

The expression vector pComb3 and the cloning vector pMD18-T/L weredigested with restriction endonucleases Sac I and Xba I. Positive cloneswere selected after ligation of gel-purified digested fragments andtransformation, and confirmed by restriction analysis. The obtainedlight chain expression vector pComb3/L and the cloning vector pMD18-T/Fdwere respectively digested with restriction endonucleases XhoI and SpeI. Fab display expression vector pComb3/Fd-gIII-L was obtained afterligation of gel-purified digested fragments, transformation, selectionand confirmation. Finally, the vector pComb3/Fd-gIII-L was cut byrestriction endonuclease enzymes SpeI and NheI to remove gIII genefragment and then circularized by T4 DNA ligase to generate a newsecretory expression vector pComb3/Fab-L. The desirable clone wasobtained after transformation, screening and restriction analysis.

1.2.3 Expression of the Fab Gene

Single colonies of correct recombinant clones were inoculated separatelyin 2 mL SB-A medium containing 100 mg/L ampicillin and grown overnightat 37° C. The second day, after transfer the culture into SB-A medium ina ratio of 1:100, the culture was maintained at 37° C. until A₆₀₀ wasbetween 0.4-0.6. Then IPTG was added to a final concentration of 1mmol/L. The culture was then maintained at 30° C. overnight. Aftercentrifugation, the bacteria cells were harvested. Then they werefreezed and thawed time and again. The supernatant was harvested aftercentrifugation for further test.

1.2.4. Sandwich ELISA Detection of the Expression of Fab

An ELISA plate was coated with goat anti-mouse IgG (10 mg/L) and thenincubated overnight at 4° C., defatted milk (50 g/L) was used to blockall the wells to prevent non-specific binding. After blocking for 1hour, the supernatants of untransformed bacteria, the non-inducedpComb3/Fd-L transformant, and the induced pComb3/Fd-L transformant wereadded. McAb HAb18 was used as positive control and PBS as blank control.Finally, goat anti-mouse IgG-HRP was added with TMB as a substrate todevelop color.

1.2.5 Detection of the Antigen Binding Activity of the ExpressionProduct

1) Indirect ELISA

The wells of a microtiter plate were coated with purified GST and aprokaryotic expression product GST-HAb18GE (a fusion of theextracellular domain of hepatoma-associated antigen HAb18G with GST).Supernatants of untransformed bacteria, non-induced transformant, andinduced transformant were detected. McAb HAb18 was used as positivecontrol and PBS as blank control.

2) Competitive ELISA

The wells of microtiter plate were coated with purified GST-HAb18GE at4° C. overnight, and the wells were blocked with 50 g/L defatted milk.Then a mixture of equal amounts of HRP-HAb18 (0.1 mg/L) and differentdilutions of the supernatant of the induced transformant were added.After incubation and washing, TMB was used to develop color. A₄₅₀ wasmeasured and the inhibition rate was calculated. % Inhibition=[(A₄₅₀ ofcontrol group−A₄₅₀ of inhibition group)/A₄₅₀ of control group]×100%.

3) Immuno-Fluorescence Staining

A cell suspension of HHCC which highly expressed antigen HAb18G wasprepared. The concentration of the HHCC suspension was adjusted to5×10⁹-1×10¹⁰ cells/mL. HAb18 Fab was diluted with horse blood serum andthen added to the HHCC suspension and incubated for 30 minutes at 4° C.Cells were washed twice, FITC conjugated rabbit anti-mouse IgG was thenadded and incubated at 4° C. for 30 minutes. After washing twice, cellswere fixed and mounted and then observed under a fluorescent microscope.In this experiment, McAb HAb18 was used as a positive control, PBS asblank control, and an anti-encephalitis virus McAb as a negativecontrol.

2 Results

2.1 Amplification of Fab Gene and Construction of the Expression Vector

The genes encoding Fd and light chain of McAb HAb18 were amplified byRT-PCR using Fd 5′ and 3′ primers for Fd and 5′ and 3′primers for lightchain, respectively. The amplified products were separated by 10 g/Lagarose gel. As expected, both products were 660 bp in length (FIG. 2).Then, the genes encoding Fd and light chain of McAb HAb18 were clonedinto pMD-18-T and sequenced. Sequence analysis showed that the sequencesof the genes encoding variable regions of heavy chain and light chain(VH and VL) were identical to those of the variable region genes ofHAb18 cloned in Example 1. C_(H1) belonged to IgG1 and C_(L) belonged toκ. The construction of the recombination vector pComb3/Fd-L is shown inFIG. 3. Restriction analysis showed that the gene fragments of interest(about 660 bp) had been inserted into the vector pComb3 in one copy(FIG. 4).

2.2 Expression and Identification of Fab

Sandwich ELISA detection showed that Fab was expressed by therecombination vector pComb3/Fd-L after IPTG induction (Table 1). Resultsfrom indirect ELISA also showed that the expressed Fab was capable ofspecifically binding with the extracellular domain ofhepatoma-associated antigen HAb18G (Table 1). The competitive ELISAfurther showed that the expressed Fab could competitively inhibit thebinding of HAb18 with its specific antigen. The inhibition ratedecreased with the decrease of the concentration of the expressedproduct (FIG. 5). The immuno-fluorescence staining revealed that theexpressed Fab could specifically bind with hepatocarcinoma cell lineHHCC (FIG. 6A), but with a fluorescent intensity weaker than that of thepositive control (FIG. 6B). No immuno-fluorescence staining was observedin negative control (not shown). TABLE 1 ELISA analysis of the Fabbinding activity (χ ± S) Results of Results of Sandwich Indirect GroupsELISA(A₄₅₀) ELISA(A₄₅₀) Transformant with empty vector* 0.075 ± 0.0040.075 ± 0.004 Transformant with pComb3/Fd-L, 0.017 ± 0.002 0.098 ± 0.008not induced* Transformant with pComb3/Fd-L, 0.633 ± 0.014^(a) 0.379 ±0.013^(a) induced HAb18 1.271 ± 0.016 1.018 ± 0.012 PBS 0.059 ± 0.030.068 ± 0.002^(a)p < 0.01, compared with transformant with empty vector;*the supernatant of the transformant culture.

Example 3 Construction of a Universal Expression Vector for a ChimericHuman/mouse Fab Antibody and Expression of the Chimeric Fab AntibodyAgainst Human Hepatoma Associated Antigen HAb18G

HAb18 McAb is a murine monoclonal antibody with high specificity andaffinity for human hepatoma associated antigen HAb18G/CD147. A murineantibody can elicit human anti-mouse antibody (HAMA) reaction todifferent extents in the body of patients. This reaction will weaken thetherapeutic efficacy of the antibody, produce toxicity and damage theorgans for elimination. In addition, owing to its poor penetratingability, the clearance of a large antibody molecule in blood andnon-tumor tissues is relatively slower, leading to the lower T/NT ratio,which limits the further application of said antibody. In order toovercome these defects, it is necessary to develop a new type ofgenetically engineered antibody with low immunogenicity and high T/NTratio. The relative molecular weight (Mr) of chimeric Fab is about50,000, making it have better penetration and pharmacokineticcharacteristics. Fabs have been used in diagnosing and treating variousdiseases. In this example, starting from the Fab antibody constructedaccording to Example 2, a partially humanized Fab antibody wasconstructed by replacing the murine mCH1/mCL with huCH1/huCL in order todecrease the HAMA response to a great extent.

1. Materials and Methods

1.1 Materials

The vector pComb3/HuFab containing human IgG3CH1 and κ light chainconstant region genes was kindly provided by Dr Wen, Department ofBiochemistry, FMMU, China. T vectors pMD18T-Fd and pMD18T-L containing,respectively, McAb HAb18 (IgG1) Fd and the entire light chain wereconstructed by the inventors. Expression vector pComb3 and E. colicompetent cells JM109 and XLI-blue were purchased. T vector, PCRreagents, restriction endonucleases and ligase were purchased fromTakara (Dalian, China). McAb HAb18, chimeric HAb18IgG and HRP-HAb18 weremade by the inventors (Zhinan Chen, Yanfang Liu, Jizheng Yang et al.McAb communications. 1989; 2:33-36). IPTG-, FITC- and HRP-labeled goatanti-mouse IgGs were purchased form SABC Co. Hepatocarcinoma cell lineHHCC was maintained and cultured by the Cell Engineering ResearchingCenter of the FMMU. Primers for PCR were synthesized by SBC (Beijing,China), with the following sequences: MVHback 5′-AAG TGA AGC TTC TCG AGTCTG G-3′ (SEQ ID NO:19) MVHfor 5′-GGG GAT ATC TGC AGA GAC AGT GAG -3′(SEQ ID NO:20) HuCH1back 5′-GGG GCT CGA GTT GAT ATC TCC ACC AAG GGC CCATCG (SEQ ID NO:21) GTC-3′ HuCH1for 5′-GCA TGT ACT AGT TTT GTC ACA AGATTT GGG -3′ (SEQ ID NO:22) MVLback5′-CAGATGTGAGCTCAGTATTGTGATGACCCAGACTCC-3′ (SEQ ID NO:23) MVLfor 5′-GGGGTC GAC GTT TTA TTT CCA ACT TTG T-3′ (SEQ ID NO:24) HuCLback 5′-GTTCCG AGC TCA AGT CGA CCT GTG GCT GCA CCA TCT (SEQ ID NO:25) GTC-3′HuCLfor 5′-GCG CCG TCT AGA ATT AAC ACT CTC CCC TGT TGA AGC TCT (SEQ IDNO:26) TTG TGA CGG GCG AAC TCA GGC CC-3′

The underlined sequences in the primers are the restriction sites of XhoI, EcoR V, Xho I, EcoR V, Spe I, Sac I, Sal I, Sac I, Sal I and Xba I,respectively.

1.2 Methods

1.2.1 Construction of the Universal Expression Vector for ChimericHuman/Mouse Fab Antibodies

The vector pComb3/HuFab was used as template to amplify the humanIgG3CH1 and κ light chain constant region genes. Primer pairs HuCH1backand HuCH1for, and HuCLback and HuCLfor were used respectively. The PCRreaction products were purified and retrieved using a gel purificationkit, followed by ligating into the vector pMD18-T to construct cloningvectors pMD18-T/HuCH1 and pMD18-T/HuCκ. After the vectors weretransformed to Escherichia coli JM109, positive clones screened byrestriction analysis were sequenced. The expression vector pComb3 andthe cloning vector pMD18-T/Hu C□ were cleaved by restrictionendonucleases SacI and XbaI. The desired fragments were separated byagarose gel electrophoresis. The pComb3/HuC□ positive clones generatedby ligation and transformation were selected and identified byrestriction. Then, the vector pComb3/HuC□ and cloning vectorpMD18-T/HuCH1 were cleaved by XhoI+SpeI and desired fragments wereligated. After ligation, transformation, selection, and identification,a chimeric Fab displaying universal vector pComb3C was constructed.

1.2.2. The Construction of the Hab 18cFab Expression Vector

The heavy chain and light chain variable region genes VH and VL of McAbHAb18 were amplified using the vectors pMD18T-Fd and pMD18T-L astemplates, respectively, with the corresponding primer pair of MVHbackand MVHfor, or MVLback and MVLfor. The corresponding restriction siteswere also incorporated simultaneously. Furthermore, SacI+SalI were usedto cleave respectively the expression vector pComb3C and the PCR productthat was amplified from the heavy chain variable region gene of mAbHAb18 when the gene was purified and retrieved by Gel Purification Kit.Through such genetic manipulations as purification of desired fragmentsby agarose gel electrophoresis, ligation, transformation, the pComb3C/cL positive clones were selected and identified by restriction. Then,the constructed pComb3 C/cL and PCR product that was amplified from thelight chain variable region gene of mAb HAb18 when the gene was purifiedand retrieved by Gel Purification Kit were cleaved by XhoI+EcoRV. Afterligation, transformation, selection and identification procedures, thechimeric Fab displaying vector pComb3C/cFab-gIII was constructed. ThegIII fragment in the correct recombinant pComb3C/cFab-gIII was cleavedby SpeI+NheI, which then circularized to a secretory expression vectorpComb3C/Fab by T4 DNA ligase. Repeating the above transformation,selection and identification by restriction, the desired clones wereobtained.

1.2.3 The Expression of the cFab Gene

An individual colony containing the correct identified recombinant clonewas seeded in 2 mL of SB-A medium supplemented with 100 mg/L ofampicillin and cultured overnight at 37□. On the next day, the culturewas seeded in fresh SB-A medium at 1:100 and cultured at 37 □ until A₆₀₀reached about 0.4-0.6. Then, IPTG was added in a final concentration of1 mmol/L and cells were cultured overnight at 37° C. Aftercentrifugation, the bacteria cells were harvested. Then they werefreezed and thawed time and again. The supernatant was harvested aftercentrifugation for further test.

1.2.4. Sandwich ELISA Detection of the Expression of cFab

An ELISA plate was coated with goat anti-human IgG (10 mg/L) and thenincubated overnight at 4° C., defatted milk (50 g/L) was used to blockall the wells to prevent non-specific binding. After blocking for 1hour, the above-mentioned supernatants of untransformed bacteria, thenon-induced pComb3/cFab transformant, and the induced pComb3/cFabtransformant were added. HAb18IgG was used as positive control and PBSas blank control. Finally, goat anti-human IgG-HRP was added with TMB asa substrate to develop color.

1.2.5 Detection of the Antigen Binding Activity of the ExpressionProduct

1) Indirect ELISA

The wells of a microtiter plate were coated with purified GST and aprokaryotic expression product GST-HAb18GE (a fusion of theextracellular domain of hepatoma-associated antigen HAb18G with GST).Supernatants of untransformed bacteria, non-induced transformant, andinduced transformant were analyzed. HAb18IgG was used as positivecontrol and PBS as blank control.

2) Competitive ELISA

The wells of a microtiter plate were coated with purified GST-HAb18GE at4° C. overnight, and the wells were blocked with 50 g/L nonfat driedmilk. Then a mixture of equal amounts of HRP-HAb18 (0.1 mg/L) anddifferent dilutions of the supernatant of the induced transformant wereadded. After incubation and washing, TMB was used to develop color. A₄₅₀was measured and the inhibition ratio was calculated. %Inhibition=[(A₄₅₀ of control group−A₄₅₀ of inhibition group)/A₄₅₀ ofcontrol group]×100%.

3) Immuno-Fluorescence Staining:

A cell suspension of HHCC which highly expressed the HAb18G antigen wasprepared. The concentration of the HHCC suspension was adjusted to5×10⁹-1×10¹⁰ cells/mL. HAb18 Fab was diluted with horse blood serum andthen added to the HHCC suspension and incubated for 30 minutes at 4° C.Cells were washed twice, FITC conjugated rabbit anti-human IgG was thenadded and incubated at 4° C. for 30 minutes. After washing twice, cellswere fixed and mounted and then observed under a fluorescent microscope.In this experiment, chimeric HAb18IgG was used as a positive control,PBS as blank control, and human IgG as a negative control.

2. Results

2.1 Construction of the Universal Expression Vector for ChimericHuman/Mouse Fab Antibody

Using two pairs of primers, HuCH1back and HuCH1for, and HuCLback andHuCLfor respectively, the desired genes, which had the expected size,were amplified successfully. After they were cloned into the T vector,sequence analysis demonstrated that the sequences of these genes werecompletely identical with the sequences of known human IgG3CH1 gene andhuman κ light chain constant region gene. These genes were respectively324 bp and 333 bp in length. Also correct restriction sites have beenincorporated. Restriction analysis of the recombinant universalexpression vector pComb3C showed that the IgG3CH1 gene and human κ lightchain constant region gene have been correctly inserted into thecorresponding restriction sites of the vector pComb3 (FIG. 7).

2.2 Construction of HAb18cFab Gene Expression Vector

Using two pairs of primers, HuCH1back and HuCH1for, and HuCLback andHuCLfor respectively, desired genes with expected size were amplified.The recombinant expression vector pComb3C/cFab was constructed throughcleavage and ligation of the vector and the corresponding genes (FIG.8). Restriction analysis showed that the desired gene fragments had beencorrectly inserted into the corresponding restriction sites of thevector pComb3C (FIG. 9). Sequence analysis showed that the heavy chainvariable region (VH) sequence in the chimeric Fd and the light chainvariable region (VL) sequence in the chimeric light chain were identicalwith the cloned sequences of the variable regions of McAb HAb18. Thecorresponding restriction sites were also adequately incorporated.Meanwhile, the murine VH and VL were respectively correctly ligated withhuman IgG3CH1 and κ light constant region through the restriction sitesof EcoR V and Sal I. The open reading frame was correct.

2.3 Induced Expression and Identification of cFab

Sandwich ELISA detection showed that cFab was expressed by therecombination vector pComb3/cFab after IPTG induction (FIG. 10). Resultsfrom indirect ELISA also showed that the expressed cFab was capable ofspecifically binding with the extracellular domain of HAb18G (FIG. 10).The competitive ELISA further showed that the expressed cFab couldcompetitively inhibit the binding of HAb18 with its specific antigen.The inhibition rate decreased with the decrease of the concentration ofthe expressed product (FIG. 11). The immuno-fluorescence stainingrevealed that the expressed cFab could specifically bind with ahepatocarcinoma cell line HHCC (FIG. 12A), but with a fluorescentintensity weaker than that of the positive control (FIG. 12B). Noimmuno-fluorescence staining was observed in a negative control (datanot shown).

1. An anti-human hepatocarcinoma monoclonal antibody HAb18 heavy chainvariable region gene, having the sequence of SEQ ID NO:1, or a sequencehybridizable with SEQ ID NO: 1 under stringent condition.
 2. Apolypeptide encoded by the gene in claim 1, comprising the sequence ofSEQ ID NO:
 3. 3. An anti-human hepatocarcinoma monoclonal antibody HAb18light chain variable region gene, having the sequence of SEQ ID NO:2, ora sequence hybridizable with SEQ ID NO:2 under stringent condition.
 4. Apolypeptide encoded by the gene in claim 3, comprising the sequence ofSEQ ID NO:
 4. 5. An expression vector comprising the gene in claim 1 orclaim
 3. 6. A host cell transformed or transfected by the expressionvector in claim
 5. 7. A recombinant Fab gene, comprising the heavy chainvariable region gene in claim 1, and the light chain variable regiongene in claim
 3. 8. An expression vector, comprising the recombinant Fabgene in claim 7, wherein the vector is pComb3/Fd-L.
 9. A recombinant Fabantibody, produced by the expression of the expression vector in claim8.
 10. A chimeric human/mouse Fab gene, comprising the heavy chainvariable region gene in claim 1, and the light chain variable regiongene in claim
 3. 11. An expression vector comprising the chimerichuman/mouse Fab gene in claim 10, wherein the vector is pComb3C/cFab.12. A chimeric human/mouse Fab antibody, produced by the expression ofthe expression vector in claim
 11. 13. Use of the gene in claim 1 or 3in the preparation of a medicament for the diagnosis or treatment ofhepatocarcinoma.